The enormous complexity of the brain is derived from hundreds of neuronal cell types and extensive synaptic connections between them. Studies of the localized function of the brain subregions have recently been revolutionized by the development of genetic engineering that ideally switches gene expression on and off in a particular cell-type of a certain brain subregion in vivo. For example, Cre recombinase of the P1 bacteriophage has proven invaluable for conditional transgenic manipulation in post-mitotic neuronal cells of the adult brain. Three years ago, we initiated a project to create a variety of brain-subregion or cell-type restricted conditional transgenic mice. Last year, we established a dentate-mossy cell Cre strain (Cre#4688), which has been crossed with floxed-NMDAR1 strain to create mossy cell-restricted NR1 KO mice. This project is described separately (#MH002869-01). To create additional subregion-restricted Cre-recombinase transgenic lines, several BAC (Bacterial Artificial Chromosome) clones carrying a promoter for directing the expression in particular brain areas were used to knock-in Cre recombinase cDNA by homologous recombination. For instance, BAC 6-Cre for forebrain interneurons, BAC 11-Cre for hippocampal CA1, BAC 14-Cre for prefrontal cortex, BAC16-Cre for VTA, and BAC 20-Cre for NAcc have been injected into eggs to generate Cre transgenic mice in the Transgenic Core Facility. Once Cre-lines are established from these founder mice, we cross them with a Rosa26 reporter line, in which the expression of Cre recombinase is visualized by X-gal staining. Most of the lab members have participated in this project. Kimberly Christian is working on the prefrontal cortex project. Zhihong Jiang is working on hippocampal CA1/subiculum project. Juan Belforte, a visiting fellow supported by NIAAA, is working on BAC clones which contain Nucleus accumbens and central catecholaminergic neurons, locus coeruleus (LC) and ventral tegmental area (VTA). Catherine Cravens and Angela Miracle are engaged in the genotyping many of these projects? lines. Kazu Nakazawa is involved in forebrain interneuron projects, as well as supervising other lab members? projects. While the screening of founders is still underway, we are in the process of establishing Cre transgenic strains, in which Cre expression is restricted to locus coeruleus and medial habenula, respectively. Once these lines are established, we will further narrow down this project to target the NMDA receptor knockout to particular cell types and investigate the behavioral and physiological consequence of region-restricted knockout of NMDA receptors (NRs). The behavioral and physiological analyses of these future NR knockout mice will hopefully lead to our understanding of the most serious neuropsychiatric disorders, such as bipolar disorders and schizophrenia. We have also initiated a floxed mouse project to explore the in vivo function of particular brain areas, that is, to generate transgenic mice in which cell ablation can be induced only in the forebrain cells where Cre recombinase is highly expressed. We use a variant of human heparin-binding-epidermal growth factor receptor (HB-EGF) precursor as a diphtheria toxin receptor (DTR). Since diphtheria toxin (DT) administered by intra-peritoneal injection does not bind to the murine HB-EGF precursor, cell ablation by DT binding will depend on the expression of human HB-EGF following Cre-loxP excision in the floxed HB-EGF alleles of mice. In collaboration with Dr. Kenji Kohno in Nara, Japan, Kazu Nakazawa, Kimberly Murdaugh, and Yoko Yabe, have engaged in making the construct of floxed-human HB-EGF. Also, we have introduced a floxed mice carrying simian HB-EGF in Rosa26, a house keeping gene locus, from Drs. Ari Waisman and Thorsten Buch in Germany. Essentially, this imported strain is for the same rationale. However, since HB-EGF expression is directed by the Rosa26 locus, this strain cannot be used when Cre recombinase is expressed in outside the brain. Nevertheless, we are crossing these floxed-DTR lines with some of our Cre lines, which show restricted expression in the brain.